A critical issue in the development of an effective HIV vaccine is the need to understand the immune correlates of protection in HIV and SIV infection and to develop and understand vaccine approaches that drive effective mucosal immune responses. Without such knowledge, it is difficult to ascertain the potential effectiveness of a vaccine. Indeed, despite promising immunogenicity, few viable vaccine candidates designed to stimulate T cell responses have proven particularly effective in controlling pathogenic SIV challenge, which may in part be due to the lack of vaccine-induced mucosal responses. Additionally, a major challenge has been the difficulty in identifying the qualitative aspects of T cell functionality most correlated with protection. Recent work in our laboratory using polychromatic flow cytometry that can simultaneously assess multiple T cell functions (degranulation, IFNg, TNFa, IL2,and MIP1b) has revealed dramatic differences in the functional profile of the T cell response to HIV in progressive and non-progressive HIV disease. Through these studies, we have found that the functional profile of the HIV-specific CD8+ T cell response itself represents a correlate of immune protection. In this program we combine our knowledge-based understanding of T cell functional characterization with novel vaccines that induce mucosal immune responses. Here, we will extend our techniques into murine and primate HIV DNAvaccine models to better understand the nature of the immune responses these vaccine candidates elicit. Our preliminary data indicates that through systemic administration of DNA vaccines and chemokine adjuvants, we can expand the mucosal component of the vaccine-induced response. Thus, we will therefore be able to define the functional, phenotypic, and protective qualities of a systemic and mucosal vaccine-induced immune response. Ultimately, such knowledge will be pivotal in the design and implementation of effective HIV vaccines designed to stimulate protective mucosal immune responses. There are three hypotheses: (1) Systemic immunization will elicit T cell responses with distinct qualitative profiles at mucosal and systemic compartments, based on multiple functions assessed simultaneously. (2) The mucosal chemokines TECK, MEC, and CTACK can be utilized as adjuvants to manipulate the functional quality and phenotype of systemic and mucosal DNA vaccine-induced T cells to enhance vaccine efficacy. (3) Chemokine adjuvants can influence mucosal trafficking of DNA vaccine-induced T cells through recruitment of new responses or alteration of the functional profiles of cells responding to the DNA vaccine itself.